In silico characterization of fructosyl peptide oxidase properties from Eupenicillium terrenum.

Fructosyl peptide oxidase (FPOX) enzyme from Eupenicillium terrenum has a high potential to be applied as a diagnostic enzyme. The aim of the present study is the characterization of FPOX from E. terrenum using different bioinformatics tools. The computational prediction of the RNA and protein secondary structures of FPOX, solubility profile in Escherichia coli, stability, domains, and functional properties were performed. In the FPOX protein, six motifs were detected. The d-amino acid oxidase motif was found as the most important motif that is a FAD-dependent oxidoreductase. The cysteines including 97, 154, 234, 280, and 360 showed a lower score than -10 that have a low possibility for participitation in the formation of the SS bond. The 56.52% of FPOX amino acids are nonpolar. Random coils are dominant in the FPOX sequence, followed by alpha-helix and extended strand. The fpox gene is capable of generating a stable RNA secondary structure (-423.90 kcal/mol) in E. coli. FPOX has a large number of hydrophobic amino acids. FPOX showed a low solubility in E. coli which has several aggregation-prone sites in its 3-D structure. According to the scores, the best mutation candidate for increasing solubility was the conversion of methionine 302 to arginine. The melting temperature of FPOX based on its amino acid sequence was 55°C to 65°C. The amounts of thermodynamic parameters for the FPOX enzyme were -137.4 kcal/mol, -3.59 kcal/(mol K), and -6.8 kcal/mol for standard folding enthalpy, heat capacity, and folding free energy, respectively. In conclusion, the in silico study of proteins can provide a valuable method for better understanding the protein properties and functions for use in our purposes.

The Agglutinin of Common Nettle (Urtica dioica L.) Plant Effects on Gene Expression Related to Apoptosis of Human Acute Myeloid Leukemia Cell Line.

Treatment of acute myeloid leukemia (AML) requires new drugs as result of a rise in new cases and high disease relapse. Plant lectins with the ability to bind carbohydrates on the cell surface have the potential to treat cancer. Urtica dioica L. agglutinin (UDA) is a low weight lectin with anti-benign prostatic hyperplasia (BPH) impact. Here, we examine the impact of UDA on HL-60 cell line. Cytotoxicity and cytostatic effects were assessed in HL-60 cells treated with UDA and vincristine (positive control). The effects of the lectin on cell cycle phases and cell death mechanism were surveyed by propidium iodide (PI) staining and annexin V/PI, respectively. The activation status of the apoptosis pathway was determined by western blotting. Finally, the expression levels of 84 genes were examined by the Human cancer drug target gene PCR array kit. The results indicated that the increase in UDA concentration inhibited the proliferation of HL-60 cells as well as apoptosis induction. Cell cycle analysis showed that the number of sub G1 cells increased essentially. Experimental observations showed that UDA can induce cell apoptosis through a caspase 9-dependent pathway. The expression changes of 21 genes confirmed the apoptotic events in HL-60 cells treated with UDA. In this, we have presented the first investigation on the cytotoxic and apoptotic effects of a lectin isolated from rhizomes and roots of Urtica dioica L. on human AML cells. Generally, the results suggest that UDA may have therapeutic value for leukemia and would be studied further as a new drug for AML later on.

Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm.

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.

Heterologous expression, purification, and refolding of SRY protein: role of L-arginine as analyzed by simulation and practical study.

Escherichia coli is a widely-used cell factory for recombinant protein production, nevertheless, high amount of produced protein is seen in aggregated form. The purpose of this study was to improve the solubility of recombinant bovine sex-determining region Y protein (rbSRY) by exploring the effect of temperature, inducer, and water-arginine mixed solvent. Codon-optimized rbSRY expressed in Rosetta-gami B (DE3) pLysS and purified by NI-NTA His-select affinity chromatography in the native and denaturing conditions. A three-dimensional model of SRY was built and studied through molecular dynamics simulations in water and in the presence of L-arginine as co-solvent. Results indicated the significant effects of temperature and IPTG concentration (P < 0.001) on the solubility of rbSRY. The binding activity of native, inclusion bodies and refolded fractions to anti-rbSRY monoclonal antibody were concentration-dependent (P < 0.001). Based on molecular modeling results, the propensity of fragments in the N-terminal domain to form ß-sheet and the relative instability of α-helices in terminal domains are the probable reasons for the high aggregation potential of SRY, which are mitigated in the presence of L-arginine. Altogether, our rbSRY protein was properly produced and applying appropriate culture conditions could help enhance its solubility, refold inclusion bodies, and improve its activity upon refolding.

Urtica dioica agglutinin (a plant lectin) has a caspase-dependent apoptosis induction effect on the acute lymphoblastic leukemia cell line.

Urtica dioica agglutinin (UDA) is a very small plant lectin with anti-prostatic activity. In this study, we investigated the effect of UDA on proliferation and apoptosis induction in human acute lymphoid leukemia (ALL) cell lines. The effect of UDA on Jurkat and Raji cell proliferation was examined by MTS assay. Distribution of cell cycle phases was determined by PI staining and apoptosis was examined with annexin V/PI and western blot. Results showed UDA treatment reduced cell proliferation in cells by inducing apoptosis. PI staining was associated with a higher percentage of the cell population in sub G1. Caspase-8 and caspase-9 dependent apoptosis occurred in Jurkat cells. Generally, UDA treatment resulted in cell death in ALL cell lines and induced apoptosis in the T-ALL cell line, Jurkat, through extrinsic and intrinsic pathways. These results may be considered as a guide to working on UDA as an anti-leukemic drug in the future.

Bacterial starter cultures induce suitable changes in milk fatty acid profiles at different fermentation conditions.

This study aimed to compare the effect of different ratios of Streptococcus thermophilus to Lactobacillus bulgaricus (3 : 1, 1 : 1, and 1 : 3) under the various stressful temperatures (37 and 45°C) on the fatty acid profiles quality of Kermanshahi roghan (yogurt by-product) and sour cream to obtain a formula for producing a kind of animal fat healthier than milk and cream. Stresses such as fermentation play an important role in bacterial behavior and consequently in food quality. Our findings presented a significant difference between roghan and sour cream fatty acid levels only at 37°C. Furthermore, starter culture 3 : 1 was the best starter for producing products with a higher quality of fatty acid profile at 37°C, and a 1 : 1 S. thermophilus to L. bulgaricus ratio was optimal at 45°C. It seems that bacteria adapt to harsh growth conditions by changing the fatty acid profiles, and these changes warrant consideration in the production of a kind of animal fat with the best fatty acid profiles. In conclusion, the roghan fatty acid profile is more suitable than sour cream only at 37°C.

2-Methylpyridine-1-ium-1-sulfonate as an Inducer of Apoptosis and Cell Cycle Arrest: A comparative in vitro and Computational Study.

"Let food be thy medicine and thy medicine be thy food" was expressed by Hippocrates and the health benefits of medicinal plants and natural products have been considered by humans since historic times. The current study aims to investigate the anti-cancer activity of 2-Methylpyridine-1-ium-1-sulfonate (MPS) isolated from bulbs of Allium hirtifolium. The MPS compound (in a dose-dependent manner) induced arrest the AGS cells in G1 and G2/M phases, and Caco-2 cells in G1 and S phases. These findings were associated with the down-regulation of cyclin D1, CDK4, and up-regulation of p21, p27 and p53. According to the morphological observations and DNA fragmentation assay, the MPS compound induced apoptosis in both cell lines, and also cause a significant increase in the expression of Bax/Bcl-2. In this context, our molecular docking results unveiled that the MPS compound has considerable affinity to interact with the minor groove of ctDNA and also with cell cycle kinases. To approve and find the accurate MPS mode of action against cancer cell lines (especially in gastrointestinal cancer) further studies is highly recommended.

In vivo anti-inflammatory efficacy of the combined Bowman-Birk trypsin inhibitor and genistein isoflavone, two biological compounds from soybean.

Soybean Bowman-Birk protease inhibitor (BBI) and genistein, two biological compounds from soybean, are well-known for their anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was designing a BBI-genistein conjugate and then investigating its protective effect on lipopolysaccharide (LPS)-induced inflammation in BALB/c mice, compared with the effects of combination of BBI and genistein. BBI was purified from soybean and the BBI-genistein conjugate was synthesized. The BALB/c mice were intraperitoneally treated 2 hours before LPS induction. Our results showed that treatment with the combination of BBI and genistein greatly led to more reduced serum levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ compared with the treatments of BBI alone, the BBI-genistein conjugate, and genistein alone, respectively. Moreover, the expression of TNF-α and IFN-γ in the splenocytes was significantly downregulated along with improving host survival against the LPS-induced lethal endotoxemia in the same way. Our data support a new combined therapy using BBI and genistein, as natural anti-inflammatory agents, to develop a new drug for inflammatory diseases.

Low, but not high, dose triptolide controls neuroinflammation and improves behavioral deficits in toxic model of multiple sclerosis by dampening of NF-κB activation and acceleration of intrinsic myelin repair.

Cuprizone (Cup) is a copper chelating agent frequently used to study factors that affect oligodendrocytes (OLGs) death and acute demyelination. Triptolide (TP), a nuclear factor-kappaB (NF-κB) blocker, is a major bioactive component of Tripterygium wilfordii Hook f. (TWHf) with various therapeutic activities. In this study, we examined the effects of TP on neuroglia activation, inflammation, apoptosis, demyelination, and behavioral deficits in the Cup-induced toxic model of multiple sclerosis (MS). C57BL/6 J mice were fed with chow containing 0.2% Cup for 6 weeks to induce detectable neuroinflammation and myelin loss. TP was administered intraperitoneally at different doses (125, 250 or 500 µg/kg/day) during the last week of the Cup challenge. Although TP substantially decreased Cup-induced NF-κB extra activation, TNF-α and IL-1 over expression, and gliosis in a dose-dependent manner, only low dose of TP (TP-125) was able to raise the number of OLGs precursor cells (NG-2+/O4+), reduce Bax/Bcl-2 ratio and improve behavioral deficits. In addition, TP-125 decreased NF-κB activation on GFAP+ astrocytes more than MAC-3+ microglial and MOG+ oligodendrocytes which suggested the possibility of specific dampening of NF-κB signaling in reactive astrocytes. Behavioral assessments by open-field and rota-rod tests showed that only TP-125 notably improved motor function and motor coordination compared to the Cup group. These findings highlight the pivotal role of NF-κB signaling in the oligodendrogenesis and lesion reduction in demyelination diseases such as MS.

Effect of planting time and vermicompost on the proteomic pattern of fenugreek (Trigonella foenum-graecum)

The fenugreek is one of the most important medicinal plants belongs to Fabaceae, originated in West Asia, Iran and Mediterranean regions. This research included a qualitative study of fenugreek proteins using SDS-PAGE electrophoresis on polyacrylamide gel and the separation of protein bands of fenugreek leaves in different treatments of vermicompost fertilizer and cultivating dates. Results showed that a band (about 80 kDa) on the first planting date (May 31) is observed in all samples except for sample a1 (10 t/ha vermicompost on May 31). Another significant difference was the band contained in the third planting date (31 September) and in the molecular weight of about 15 kDa, which was not seen in other dates. This difference can be due to the synthesis of this protein with the mentioned weight under the conditions of reducing the temperature in the early fall. It also showed more differences in two-dimensional electrophoresis, for example, in 14 kDa and PI in the range of 4.5-4.7 in treatment without fertilizer, no protein expression was observed, which was consistent with the results of the SDS-PAGE test.

Anti-angiogenesis effect of ß-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive properties under experimental model.

Angiogenesis is a process through which new capillaries are formed from pre-existing ones, which contributes significantly to the pathogenesis of numerous diseases, such as cancer and chronic inflammatory disorders. The ß-D-mannuronic acid (M2000) is a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive effects and is a matrix metalloproteinase (MMP) inhibitor. This research aimed to study the anti-angiogenesis effects of M2000 under in vitro and in vivo models. The cytotoxic and anti-proliferative effects of M2000 were examined using the trypan blue method and the MTT assay, respectively. The 3D collagen-cytodex model and the chick chorioallantoic membrane (CAM) assay were then used to evaluate the anti-angiogenesis property of M2000. Cytotoxicity assay revealed that M2000 (at concentrations of less than 100 µg/mL) had no cytotoxic effect on human umbilical vein endothelial cells (HUVECs). It was also illustrated that M2000 had little or no anti-proliferative effect on HUVECs. In addition, the anti-angiogenesis effects of M2000 were shown to be marginal in the in vitro model and both significant and dose-dependent in the in vivo status. This study showed that M2000 could be considered as an anti-angiogenic molecule which more likely exerts its activity mainly via indirect effects on endothelial cells and its anti-inflammatory effects may partly be attributable to its anti-angiogenic activity. Therefore, it could be recommended as a candidate for prevention and treatment of cancer, chronic inflammatory diseases, and other angiogenesis-related disorders.

Effect of fermentation temperature and different Streptococcus thermophilus to Lactobacillus bulgaricus ratios on Kermanshahi roghan and yoghurt fatty acid profiles.

The objective of the study reported in this Research Communication was to investigate the effect of fermentation temperature (37 and 45 °C) and different ratios of Streptococcus thermophilus to Lactobacillus bulgaricus (3 : 1, 1 : 1 and 1 : 3) on Kermanshahi roghan and yoghurt fatty acid profiles (FAP) in order to obtain a product with optimized fatty acid profiles. Kermanshahi roghan is a yoghurt by-product in western Iran (Kermanshah). The results revealed that incubation temperature at 37 °C as compared to 45 °C had a better effect on fatty acid profiles of roghan and yoghurt. Furthermore, the results showed that fatty acid profile of roghan is better than yoghurt at two experimental temperatures. On the other hand, the roghan products made by equal ratio of S. thermophilus and L. bulgaricus (1 : 1) had the best quality of fatty acid profiles. Although a lower incubation temperature increases incubation time, our finding suggests that inoculation ratio 1 : 1 at 37 °C as compared to 45 °C can affect the quality of roghan and yoghurt fatty acid profiles.

Bovine Sex Determining Region Y: Cloning, Optimized Expression, and Purification.

Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.

Neuroprotective effects of ellagic acid on cuprizone-induced acute demyelination through limitation of microgliosis, adjustment of CXCL12/IL-17/IL-11 axis and restriction of mature oligodendrocytes apoptosis.

CONTEXT: Ellagic acid (EA) is a natural phenol antioxidant with various therapeutic activities. However, the efficacy of EA has not been examined in neuropathologic conditions. OBJECTIVE: In vivo neuroprotective effects of EA on cuprizone (cup)-induced demyelination were evaluated. MATERIAL AND METHODS: C57BL/6 J mice were fed with chow containing 0.2% cup for 4 weeks to induce oligodendrocytes (OLGs) depletion predominantly in the corpus callosum (CC). EA was administered at different doses (40 or 80 mg/kg body weight/day/i.p.) from the first day of cup diet. Oligodendrocytes apoptosis [TUNEL assay and myelin oligodendrocyte glycoprotein (MOG+)/caspase-3+ cells), gliosis (H&E staining, glial fibrillary acidic protein (GFAP+) and macrophage-3 (Mac-3+) cells) and inflammatory markers (interleukin 17 (IL-17), interleukin 11 (IL-11) and stromal cell-derived factor 1 α (SDF-1α) or CXCL12] during cup intoxication were examined. RESULTS: High dose of EA (EA-80) increased mature oligodendrocytes population (MOG+ cells, p < 0.001), and decreased apoptosis (p < 0.05) compared with the cup mice. Treatment with both EA doses did not show any considerable effects on the expression of CXCL12, but significantly down-regulated the expression of IL-17 and up-regulated the expression of IL-11 in mRNA levels compared with the cup mice. Only treatment with EA-80 significantly decreased the population of active macrophage (MAC-3+ cells, p < 0.001) but not reactive astrocytes (GFAP+ cells) compared with the cup mice. DISCUSSION AND CONCLUSION: In this model, EA-80 effectively reduces lesions via reduction of neuroinflammation and toxic effects of cup on mature OLGs. EA is a suitable therapeutic agent for moderate brain damage in neurodegenerative diseases such as multiple sclerosis.

In vitro inhibition of angiogenesis by heat and low pH stable hydroalcoholic extract of Peganum harmala seeds via inhibition of cell proliferation and suppression of VEGF secretion.

CONTEXT: Progression of cancer cells is completely dependent on its angiogenesis. Inhibition of tumor angiogenesis has shed new light on cancer treatment. As a result, anti-angiogenesis therapy represents one of the most significant advances in clinical oncology. Peganum harmala L. (Zygophyllaceae) is a native plant from the eastern Iranian region, which is used as a traditional folk medicine. Although some biological properties of this plant are determined, its effect on angiogenesis is still unclear. OBJECTIVE: We investigated the anti-angiogenic effects of heat and low pH stable hydroalcoholic extract of P. harmala seeds on endothelial cells (ECs) proliferation and VEGF secretion. MATERIALS AND METHODS: Dried Peganum seeds were purchased from Kermanshah Traditional Bazar in 2011. Hydroalcoholic extract of dried seeds (0, 10, 20, 40, 60, 80, 100, 120, and 150 µg/ml) was used for in vitro evaluation of its cytotoxicity, anti-proliferative, and anti-angiogenic effects on ECs. In vitro effect of the extract on VEGF secretion was assayed using ELISA. RESULTS: Treatment with hydroalcoholic extract at seven different concentrations resulted in significant decrease of ECs proliferation and angiogenesis with an ID50 of ∼ 85 µg/ml. VEGF secretion was (inhibited) decreased by the extracts at concentrations higher than 10 µg/ml. DISCUSSION AND CONCLUSION: Herbal plant extracts still attract attention owing to their fewer side effects comparing to synthetic drug agents. Current study indicated that hydroalcoholic extract of P. harmala seeds contains a potent anti-angiogenic component, which exerts its inhibitory effect mainly through down-regulation of essential mediators such as VEGF.

Anti-inflammatory effect of chymotrypsin to autoimmune response against CNS is dose-dependent.

Multiple sclerosis is an inflammatory autoimmune disease of central nervous system (CNS) in which inflammatory cells release pro-inflammatory cytokines, proteases, and other toxic mediators. Proteases are involved in many aspects of inflammatory process. There are many reports regarding the effect of proteases on inflammation. Chymotrypsin is a serine protease with anti-inflammatory effect. We investigated chymotrypsin effect on experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Intra-CSF injection with 0.1 mg/ml, 0.2 mg/ml chymotrypsin, or saline was done on day 7 after EAE induction. Our study demonstrated that 0.1 mg/ml chymotrypsin treatment did not decrease clinical signs, but 0.2 mg/ml chymotrypsin ameliorated clinical signs and manipulated immune response in both brain and spinal cord. Administration of 0.1 mg/ml or 0.2 mg/ml chymotrypsin led to decreased IL-17 along with increased IL-4 and FoxP3 in 0.2 mg/ml chymotrypsin-treated animals. Presumably, chymotrypsin acts in a dose-dependent manner and concentrations of chymotrypsin more than 0.2 mg/ml may have more beneficial effect.

A proteomic analysis of human follicular fluid: comparison between younger and older women with normal FSH levels.

The follicular fluid (FF) is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. Age-related infertility is usually considered as a problem that can be solved by assisted reproduction technology. Therefore, the identification of novel biomarkers that are linked to reproductive aging is the subject of this study. FF was obtained from healthy younger (20-32 years old) and older (38-42 years old) women undergoing intracytoplasmic sperm injection (ICSI) due to male factor infertility. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). The power of two-dimensional gel electrophoresis and the identification of proteins were exploited using matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry. Twenty three protein spots showed reproducible and significant changes in the aged compared to the young group. Of these, 19 protein spots could be identified using MALDI-TOF-TOF-MS. As a result of MASCOT search, five unique downregulated proteins were identified in the older group. These were identified as serotransferrin, hemopexin precursor, complement C3, C4 and kininogen. A number of protein markers were found that may help develop diagnostic methods of infertility.

Investigating efficacy of colchicine plus phenolic monoterpenes fraction as a potential treatment for patients diagnosed with COVID-19: A randomized controlled parallel clinical trial.

Background: COVID-19 now is a serious concern for the world healthcare system. This study aimed to investigate possible therapeutic effect of colchicine and phenolic monoterpenes accompanied by standard care of treatment (SCT) in patients diagnosed with COVID-19. Methods: In this randomized controlled parallel clinical trial, a total number of 179 (of 200) patients with confirmed COVID-19 were enrolled according to the inclusion and exclusion criteria. The patients were allocated by simple randomization method into two groups control (receiving SCT with 71 patients) and intervention (receiving SCT plus colchicine and phenolic monoterpenes with 107 patients). The mortality ratio during hospitalization as well as a 2-week follow-up, ICU admission rate, and hospitalization duration were assessed as main outcomes. Results: The mortality ratio was 0.9% (1/108) and 8.45% (6/71) in the intervention and the control groups (p-value = 0.035) respectively, these ratios after a 14-day follow-up were 1.85% (2/108), and 9.85 (7/71) respectively (p-value = 0.031). Also, the ICU admission was significantly lower (p-value = 0.006) in the intervention group 2/108 (1.85%) compared with controls 10/71 (14.08%). Moreover, the duration of hospitalization followed a similar pattern to ICU admission with 4.17 ± 1.34 vs. 6.39 ± 2.59 days in the intervention and control groups respectively (p-value< 0.001). Furthermore, no significant side effect was found between the groups. Conclusion: According to the results, the combination of colchicine plus phenolic monoterpenes could be an additive treatment for the SCT. The authors strongly recommend further trials on this combination with other SCTs.

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli.

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.

Genetic diversity study of some medicinal plant accessions belong to Apiaceae family based on seed storage proteins patterns.

Cumin (Cuminum cyminum L.), Fennel (Foeniculum vulgare L.) and Longleaf (Falcaria vulgaris Bernh) that all belong to Apiaceae family as medicinal plants are very important in many countries. Study of genetic diversity for medicinal plant is important for researches in future. One of the methods to evaluate plant genetic diversity and classification of them is the electrophoresis of seed storage proteins. This research was conducted in order to evaluate seed protein variability in different Iranian Cumin, Fennel and Longleaf accessions and grouping them based on these proteins as a biochemical marker. For this purpose, the samples were first powdered in liquid nitrogen and seed protein was extracted with extraction buffer. Then total soluble proteins were resolved on 12.5 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. The electrophoretic protein pattern showed 38 bands that were low polymorphism among the accessions. The result of cluster analysis showed that the accessions were classified in three groups (all 29 Cumin accessions in the first group, three Fennel ecotypes in second group and three Longleaf accessions in the last one).